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Decay of Beta-Globin mRNA in Erythroid Cells

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2014, Doctor of Philosophy, Ohio State University, Biomedical Sciences.
mRNAs targeted by endonuclease decay generally disappear without detectable decay intermediates. Nonsense-containing human Beta-globin mRNA is a unique exception. Detectable 5’truncated Beta-globin RNAs are stabilized in the cytoplasm of erythroid cells of transgenic mice and in transfected erythroid cell lines. A quantitative assay was developed to study the relationship of the truncated RNAs to the full-length mRNA. Addtionally, an inducible erythroid cell system was developed to allow for kinetic analysis of a precursor-product relationship. Results verified that a 5’truncated Beta-globin RNA is indeed a metastable decay product of the full-length transcript. The inducible cells allowed for knockdown to occur prior to expression of nonsense Beta-globin mRNA. Upf1 is the master regulator of NMD and its knockdown revealed an increase in full-length ß-globin and a decrease in the truncated RNA, confirming involvement of NMD. SMG6 has been identified as an endonuclease of the NMD pathway, thus SMG6 knockdown was performed prior to inducing nonsense-containing Beta-globin mRNA expression. Full-length nonsense-containing Beta-globin was increased during SMG6 knockdowm while the truncated species was decreased; which is consistent with SMG6 being involved in their generation. Next, endogenous SMG6 was knocked down and complemented back with siRNA-resistant constructs, either wild type or a SMG6 mutant with an inactive endonuclease activity. Complementation with wild type SMG6 was able to rescue the increased accumulation of the truncated RNA with the associated decrease in full-length transcript. The inactivated enzyme was unable to reverse the effects of SMG6 knockdown and the full-length mRNA was stabilized and the amount of the shortened RNA decreased. These results demonstrate that the endonuclease activity of SMG6 is responsible for the generation of the 5’truncated decay products. Notably, none of the SMG6 manipulations altered the phosphorylation state of Upf1. As a whole, these data provide the first proof for generation of stable NMD decay products by SMG6 endonuclease cleavage.
Daniel Schoenberg, PhD (Advisor)
Juan Alfonzo, PhD (Committee Member)
Dawn Chandler, PhD (Committee Member)
Denis Guttridge, PhD (Committee Member)
133 p.

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Citations

  • Dougherty, J. A. (2014). Decay of Beta-Globin mRNA in Erythroid Cells [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397499022

    APA Style (7th edition)

  • Dougherty, Julie. Decay of Beta-Globin mRNA in Erythroid Cells. 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1397499022.

    MLA Style (8th edition)

  • Dougherty, Julie. "Decay of Beta-Globin mRNA in Erythroid Cells." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397499022

    Chicago Manual of Style (17th edition)